U.S. patent application number 10/530598 was filed with the patent office on 2006-11-16 for absorption of fat-soluble nutrients.
Invention is credited to Moti Harel, David J. Kyle, John Piechocki.
Application Number | 20060258623 10/530598 |
Document ID | / |
Family ID | 33539183 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060258623 |
Kind Code |
A1 |
Harel; Moti ; et
al. |
November 16, 2006 |
Absorption of fat-soluble nutrients
Abstract
Carotenoids are provided as dietary supplements to animal feed.
These supplements improve the bioavailability of carotenoids by
providing them in combination with phospholipids. The invention
provides animal feeds for aquatic and terrestrial animals, and
methods for making the feeds.
Inventors: |
Harel; Moti; (Baltimore,
MD) ; Piechocki; John; (Odenton, MD) ; Kyle;
David J.; (Catonsville, MD) |
Correspondence
Address: |
DUANE MORRIS, LLP;IP DEPARTMENT
30 SOUTH 17TH STREET
PHILADELPHIA
PA
19103-4196
US
|
Family ID: |
33539183 |
Appl. No.: |
10/530598 |
Filed: |
June 21, 2004 |
PCT Filed: |
June 21, 2004 |
PCT NO: |
PCT/US04/19972 |
371 Date: |
November 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60479507 |
Jun 19, 2003 |
|
|
|
Current U.S.
Class: |
514/78 ; 424/442;
514/690; 514/763 |
Current CPC
Class: |
A61K 31/065 20130101;
A61K 45/06 20130101; A23K 50/80 20160501; A61K 31/66 20130101; A23K
20/22 20160501; A61K 31/065 20130101; A23K 20/26 20160501; A23K
50/75 20160501; A23K 20/179 20160501; A61K 31/685 20130101; A23K
20/158 20160501; A23K 20/174 20160501; A61K 31/66 20130101; A23K
40/30 20160501; A61P 3/02 20180101; A61K 2300/00 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/078 ;
514/690; 514/763; 424/442 |
International
Class: |
A61K 31/685 20060101
A61K031/685; A23K 1/165 20060101 A23K001/165; A61K 31/12 20060101
A61K031/12; A61K 31/015 20060101 A61K031/015 |
Claims
1-34. (canceled)
35. An animal feed composition comprising, in admixture, at least
1%, by weight, of a carotenoid and at least 5%, by weight, of a
phospholipid that is liquid at the body temperature of the
animal.
36. The composition of claim 35, wherein the ratio of the
carotenoid:the phospholipid is from 1:100 to 100:1.
37. The composition of claim 35, wherein the ratio of the
carotenoid:the phospholipid is from 1:10 to 1:1.
38. The composition of claim 35, wherein the carotenoid is obtained
from a microbial source.
39. The composition of claim 38, wherein the source is an organism
selected from the group consisting of Phaffia, Haematococcus,
Schizochytrium, and Paracoccus species.
40. The composition of claim 35, wherein the carotenoid is selected
from the group consisting of astaxanthin, zeaxanthin,
canthaxanthin, lutein, beta-carotene, and lycopene.
41. The composition of claim 35, wherein the carotenoid is a
synthetic carotenoid.
42. The composition of claim 35, wherein at least 20% of the fatty
acid residues of the phospholipid are polyunsaturated.
43. The composition of claim 35, wherein at least 10% of the fatty
acid residues of the phospholipid have three or more double
bonds.
44. The composition of claim 35, wherein at least 10% of the fatty
acid residues of the phospholipid have four or more double
bonds.
45. The composition of claim 35, wherein at least 20% of the fatty
acid residues of the phospholipid have four or more double
bonds.
46. The composition of claim 35, wherein the phospholipid is
obtained from a microbial source.
47. The composition of claim 35, wherein the phospholipid is an egg
lecithin.
48. The composition of claim 35, wherein the phospholipid is
obtained from a source selected from the group consisting of fish,
crustaceans, and shellfish.
49. The composition of claim 35, wherein the phospholipid is
obtained from a mammal.
50. The composition of claim 49, wherein the phospholipid is
obtained from a mammalian brain.
51. The composition of claim 35, wherein the animal is an aquatic
animal.
52. The composition of claim 35, wherein the animal is a fish.
53. The composition of claim 35, wherein the animal is a
crustacean.
54. The composition of claim 35, wherein the animal is a
terrestrial animal.
55. The composition of claim 35, wherein the animal is a bird.
56. The composition of claim 35, wherein the body temperature is
less than 20.degree. C.
57. A method of pigmenting an animal or an animal-derived product,
the method comprising feeding to the animal a composition
comprising, in admixture, at least 1%, by weight, of a carotenoid
and at least 5%, by weight, of a phospholipid that is liquid at the
body temperature of the animal, whereby the animal or product
becomes pigmented.
58. The method of claim 57, wherein the item that is pigmented is
selected from the group consisting of an egg and a processed egg
product.
59. The method of claim 57, wherein the item that is pigmented is
selected from the group consisting of a whole animal, the processed
flesh of an animal, and a processed animal product.
60. A method of preparing an animal feed composition, the method
comprising (a) mixing a carotenoid and a phospholipid that is
liquid at the body temperature of the animal to form a mixture; and
(b) thereafter combining the mixture with at least one other animal
feed component, such that the carotenoid makes up at least 1%, by
weight, of the composition and the phospholipid makes up at least
5%, by weight, of the composition.
61. The method of claim 60, wherein the mixture is incorporated
into a pelleted feed composition.
62. The method of claim 60, wherein the mixture is used to coat a
pelleted feed composition.
63. The method of claim 60, wherein the mixture is incorporated
into an oil used to coat a pelleted feed composition.
64. The method of claim 60, wherein multiple carotenoids are
incorporated into the mixture, the combined carotenoids make up at
least 1%, by weight, of the composition.
Description
BACKGROUND OF THE INVENTION
[0001] This application is related to improving the bioavailability
of carotenoids as provided in formulated mixtures to animals. The
invention provides both a specific composition and a method of
manufacture for improved delivery of carotenoids.
[0002] This invention relates to a carotenoid composition and
methods for its manufacture and use. In one aspect, the invention
relates to carotenoids, synthetic or naturally produced by a
single-celled organism, and phospholipids containing highly
unsaturated fatty acids. In another aspect, the invention relates
to methods of increasing carotenoid stability during feed
processing and improving bioavailability in the gastrointestinal
(GI) tract of coldwater species. In yet another aspect, the
invention relates to using products made from these carotenoid
compositions as a dietary supplement in various animal feeds.
[0003] The carotenoids, as a class of compounds, are classified
into two main groups: carotenes and xanthophylls. In contrast to
carotenes, which are pure polyene hydrocarbons, such as
beta-carotene or lycopene, xanthophylls contain oxygen functional
groups, such as hydroxyls, epoxy and/or oxo groups. Typical
representatives of the xanthophyll group are astaxanthin,
canthaxanthin and zeaxanthin.
[0004] A distinct red color is of prime importance to customer
acceptance of a subset of food products, particularly aquatic food
animals such as salmon, trout, shrimp, lobster and many other
marine animals (Hinostroza, Huberman et al. 1997; Bjerkeng and
Berge 2000). The oxygenated carotenoids (xanthophylls) are
responsible for the red color of these aquatic animals. These
xanthophylls are also useful for adding pigmentation to the flesh
and products of other animals, and to other foodstuffs, for example
poultry and eggs, various dairy products, snack foods, and the
like.
[0005] Astaxanthin is the most abundant carotenoid present in the
aquatic world (Shahidi, Metusalach et al. 1998). Aquatic animals,
like terrestrial animals, generally cannot synthesize astaxanthin
or any other carotenoid, although many of these animals accumulate
carotenoid compounds that are present in their diets. Some of these
animals, such as crustaceans, can interconvert some carotenes to
xanthophylls, of which astaxanthin is the predominant compound
formed. However, aquatic fish accumulate dietary astaxanthin even
though these fish cannot convert any other carotenoid compound to
astaxanthin. Therefore, the astaxanthin present in aquatic fish,
and in products produced from these fish, must be derived directly
from dietary sources.
[0006] Currently, synthetic astaxanthin is added to feeds of
aquacultured salmonids to provide a source of this carotenoid
(Bell, McEvoy et al. 1998). In some cases, synthetic canthaxanthin
(another xanthophyll that is very closely related to astaxanthin)
is used in place of astaxanthin in feeds for salmonids, but this
compound does not function as well in these fishes as the naturally
predominant astaxanthin (Bell, McEvoy et al. 1998).
[0007] Natural sources of dietary astaxanthin, including krill,
crawfish, crustacean processing by-products, bacteria, yeast,
algae, and higher plants are in great demand by aquacultural
industries. However, these natural sources tend to be too expensive
and of limited availability and reliability to be commercially
viable. Lycopene is an alternative natural carotenoid that might
meet the cost criterion for inclusion in feeds (Clark, Yao et al.
2000). It is in a class of carotenoids that characteristically
gives color to many vegetables.
[0008] Carotenoids are easily isomerized by heat, acid or light.
Once isomerized, they lose their biological antioxidant properties
(Fennema 1996). The high demands placed on xanthophyll-containing
formulations with respect to coloring action and bioavailability
can thus not always be met because of these problems (Yeum and
Russell 2002). Indeed, various processes and a number of combined
emulsifying/spray-drying processes (see patents DE-A-12 11 911 or
in EP-A-0 410 236) have been proposed to improve the color yields
and to increase the absorbability or bioavailability
carotenoids.
[0009] One specific problem which has not yet been addressed is
related to the low body temperature of salmonid fishes, which is
equal to the temperature of the water in which they inhabit,
generally 0 to 14.degree. C. Natural astaxanthin, especially those
in Phaffia yeasts, are concentrated in oil droplets that contain
about 13% palmitic acid (16:0) with a melting point of 64.degree.
C., and about 32% oleic acid (18:1n9) with a melting point of
16.degree. C. (Deuel 1951). Because of these high melting point
fatty acids, the astaxanthin containing oil droplets solidify near
10.degree. C. This makes it difficult for the fish to incorporate
the astaxanthin from the solidified oil droplet at water
temperatures below 10.degree. C. This is especially problematic for
coldwater fish.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention alleviates these problems by providing a
process for preparing a mixture of carotenoids and phospholipids
rich in highly unsaturated fatty acids (PUFA). The process
comprises the following steps:
[0011] a) Preparing a molecularly-associated composition of
carotenoids and a phospholipid with an edible oil or a mixture of
water and a water-miscible organic solvent. If appropriate, a
water-dispersible dry powder could also be prepared. To achieve
dispersion, e.g., in the form of a suspension or an emulsion, it is
advantageous to use an edible oil (such as, but not limited to,
sesame oil, corn oil, cottonseed oil, soybean oil, or peanut oil)
plus esters of medium chain-lengths vegetable fatty acids or fish
oils (such as, but not limited to, mackerel, capelin, menhaden or
cod liver oil).
[0012] b) Further increasing the stability of the carotenoids to
oxidative decay by adding stabilizers such as, but not limited to,
alpha-tocopherol, t-butylated hydroxytoluene, t-butylated
hydroxyanisole, ascorbic acid or ethoxyquin.
[0013] c) Providing the carotenoids used to produce the composition
from natural sources and/or synthetic sources.
[0014] d) The phospholipids used to produce the composition are
rich in polyunsaturated fatty acids (PUFA) having two or more
double bonds in at least 20% of total fatty acids.
[0015] e) The carotenoid composition according to the invention can
also contain at least one other active substance in concentrations
of 0.01 to 40% by weight.
[0016] Possible examples of these active substances are the
following:
[0017] Other carotenoids such as for example bixin, zeaxanthin,
cryptoxanthin, citranaxantin, canthaxanthin, astaxanthin,
beta-apo-4-carotenal, beta-apo-8-carotenal, beta-apo-8-carotenoic
esters, lycopene, or lutein, singly or as a mixture.
[0018] Vitamins, such as vitamin A, vitamin A acetate, vitamin A
palmitate, riboflavin, vitamin B.sub.12, ascorbic acid, ascorbyl
palmitate, nicotinic acid, nicotinamide, pyridoxine hydrochloride,
vitamin D.sub.3, tocopherol, tocopherol acetate, tocopherol
palmitate, tocotrienol, vitamin K, thiamine, calcium pantothenate,
biotin, lipoic acid, folic acid, and folic acid derivatives (such
as tetraBASF hydrofolic acid, 5-methyltetrahydrofolic acid,
10-formyltetrahydrofolic acid) and 5-formyltetrahydrofolic
acid).
[0019] Compounds with vitamin or coenzyme characteristics, such as
choline chloride, carnitine, taurine, creatine, ubiquinones,
S-methylmethionine, and S-adenosylmethionine.
[0020] Polyunsaturated fatty acids, such as linoleic acid,
linolenic acid, arachidonic acid (ARA), eicosapentaenoic acid
(EPA), and docosahexaenoic acid (DHA) and esters thereof including
but not limited to triglycerides.
[0021] Glutathione and its esters such as, for example GSH
monomethyl ester, GSH dimethyl ester, GSH monoethyl ester, and GSH
diethyl ester.
[0022] Depending on the nature of the formulation, it may contain,
besides the carotenoids, at least one other additive such as, for
example, oils, protective colloids, alkaloids (such as peperine
(Badmaev, Majeed et al. 1999)), and antioxidants.
[0023] Examples of protective colloids that can be used are
gelatin, fish gelatin, starch, dextrin, plant proteins, pectin, gum
arabic, casein, caseinate, or mixtures thereof. It is also possible
to employ polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose,
carboxymethylcellulose, hydroxypropylcellulose, and alginates.
[0024] To increase the mechanical stability of the dry powder, it
is also possible to add to the colloid a plasticizer such as sugars
or sugar alcohols, such as sucrose, glucose, lactose, invert sugar,
sorbitol, mannitol, or glycerol.
[0025] The use of the PUFA-rich phospholipids as part of this
formulation also provides additional benefit to the survival and
health of the animal consuming the invention's formulation (Bracco
and Decekbaum 1992; Furuita, Takeuchi et al. 1998; Place and Harel
2002).
[0026] The present invention provides a mixture comprising a
carotenoid and PUFA-rich phospholipid.
[0027] The present invention provides a composition comprising a
mixture including a carotenoid either in synthetic or natural form
and a phospholipid having at least 20% PUFA, where the phospholipid
is in an amount sufficient to improve carotenoid stability and
bioavailability and prevent solidification when the composition is
fed to coldwater species, and the carotenoid is in an amount
sufficient to produce acceptable coloring in edible tissues.
[0028] The present invention also provides a molecularly-associated
complex comprising a carotenoids and a phospholipid.
[0029] The present invention provides a composition comprising a
molecularly-associated complex including an amount of a carotenoid
and an amount of a phospholipid, wherein the amount of the
phospholipid is sufficient to improve carotenoids stability and
bioavailability and prevent solidification when the composition is
fed to coldwater species and the amount of the carotenoid is
sufficient to produce acceptable coloring of edible tissues.
[0030] The present invention also provides a mixture comprising a
carotenoid, a phospholipid, and a bioactive compound, or a
bioactive complex (comprising a carotenoid/phospholipid/bioactive
compound), and/or mixtures or combinations thereof.
[0031] The present invention provides a composition comprising a
mixture including a carotenoid, a phospholipid and a bioactive
compound, a bioactive complex, or mixtures or combinations thereof,
wherein the phospholipid is present in an amount sufficient to
improve the carotenoids' stability and bioavailability and prevent
solidification when the composition is fed to coldwater species,
and wherein the amount of the total carotenoid is sufficient to
produce acceptable coloring of edible tissues.
[0032] The present invention provides a composition comprising a
cellular material and a phospholipids wherein the phospholipid to
cellular material is in the ratio of from about 1:1 to about 1:100
and the cellular material comprises long chain polyunsaturated
fatty acids and/or carotenoids.
[0033] The present invention also provides a method for making a
carotenoid-containing composition with increased carotenoid
stability and bioavailability with low melting temperature when fed
to cold-water species, including the step of mixing carotenoids and
a PUFA-rich phospholipid. The method can further include the step
of mixing the carotenoid/phospholipid composition with another
bioactive compound forming an alternative and useful
composition.
[0034] The present invention also provides a method for making a
carotenoid-containing composition with increased stability and
bioavailability including the step of contacting a carotenoid and a
phospholipid under conditions sufficient to maintain the carotenoid
and the phospholipid in a molecularly-associated form. The method
can further include the step of admixing the
carotenoid/phospholipid molecular association with a bioactive
compound.
[0035] The present invention also provides for making a long chain
polyunsaturated fatty acid (LC-PUFA) composition with increased
stability and bioavailability including the step of contacting a
cellular material containing said LC-PUFA and a phospholipid under
conditions sufficient to maintain the LC-PUFA and the phospholipid
in a molecular association form. The method can further include the
step of admixing the LC-PUFA/phospholipid molecular association
with a bioactive compound.
[0036] The present invention also provides a method for enhancing
the pigmentation of coldwater animals by providing such animals
with a feed enriched with a composition that consists of a cellular
source of carotenoid such as, but not limited to Phaffia yeast,
Haematococcus algae, marigold flowers, mixed with a PUFA-enriched
phospholipid such as, but not limited to, plant lecithins, egg yolk
lecithin, phospholipid-rich extracts from animals or animal
byproducts, and phospholipid-rich extracts from microbial sources.
The cellular or synthetic carotenoid material and phospholipid
material are premixed and homogenized prior to the addition to a
feed in order to stabilize and solubilize the carotenoid and such a
process surprisingly results in the enhanced bioavailability of the
carotenoids by the coldwater animal.
BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS
[0037] FIG. 1. Improved total carotenoid content of rainbow trout
using conditions as described in Example 5 (for the Astaxanthin
compared to Astaxanthin+DHA-phospholipid) and Example 4 for
Astaxanthin compared to Astaxanthin +soy lecithin. The control had
no added astaxanthin in the diet (some residual carotenoids were in
the original diet). The soy lecithin gave a 34% higher
incorporation of astaxanthin (AX) than AX alone. The DHA-rich
phospholipid gave 56% higher incorporation of AX than AX alone.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Definitions
[0039] Unless otherwise stated, the following terms shall have the
following meanings:
[0040] The term "solution" means a liquid and any mixture of a
liquid and a solid that has fluid attributes, e.g., flowable or
having appreciable fluidity at standard temperature and pressure,
including, without limitation, a dispersion of a solid(s) in a
liquid, an emulsion, a slurry, a micro-emulsion, colloidal
suspension, a suspension, or the like.
[0041] An "emulsion" is suspension of one liquid in another with
which the first will not mix. The first liquid can be suspended as
small globules in the second liquid. An oil or an aqueous form of
the compositions of this invention can be emulsified into an
aqueous solution.
[0042] An "active substance" is any material that functions or is
capable of functioning in a manner characteristic of that
substance.
[0043] The term "molecular association" or "molecularly-associated"
means a combination of two or more molecular species associated via
any known stabilizing atomic or molecular level interaction or any
combination thereof, where the interactions include, without
limitation, bonding interactions such as covalent bonding, ionic
bonding, hydrogen bonding, coordinate bonding, or any other
molecular bonding interaction, electrostatic interactions, a polar
or hydrophobic interactions, or any other classical or quantum
mechanical stabilizing atomic or molecular interaction.
[0044] The term "species" is defined as any species in the animal
kingdom, including mammals, fish, crustaceans and mollusks.
[0045] An "aquatic animal" is an animal that lives primarily in an
aquatic environment, and includes fish, crustaceans, and mollusks.
Aquaculture methods and/or commercial production practices have
been developed to cultivate aquatic animals.
[0046] A "fish" and the plural "fish" are defined in this invention
as any Ostiechthyean or Chondrichthyean fish, such as, but not
limited to, sharks, rays, sturgeon, eels, anchovy, herring, carp,
smelt, salmon, trout, hakes, cod, rockfish, bass, drum, mackerel,
tuna, butterfish, catfish, flounder, and seabream.
[0047] A "crustacean" and the plural "crustaceans" are defined in
this invention as any member of the Class Crustacea, such as, but
not limited to, shrimp, lobsters, red claws, and crabs.
[0048] A "terrestrial animal" is one that lives primarily on land
in a non-aquatic environment, such as, but not limited to cows,
pigs, and chickens.
[0049] The term "phospholipid" refers to any lipid or fatty acid
having a covalently attached a phosphate group in the molecular
structure. These phospholipids are preferably sourced from
vegetable material such as, but not limited to, soy, corn, palm,
canola, rice, flax, coconut, combinations thereof, and are usually
obtained as byproduct of the process of refining the vegetable oil.
These phospholipids may be comprised of any of phosphatidyl choline
(PC), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE)
and/or phosphatidyl inositol (PI), or a combination thereof.
[0050] The term "PUFA-rich phospholipid" means a phospholipid
containing at least 20% fatty acids with 2 or more double
bonds.
[0051] The term "carotenoid" encompasses any molecule in a class of
yellow to red pigments, including carotenes and xanthophylls.
"Carotenes" are orange-yellow to red pigments that are found in
some animal tissues and plants, and may be converted to Vitamin A
in the liver. "Xanthophylls" are yellow pigments, some of which may
be found with chlorophyll in green plants.
[0052] Description
[0053] The inventors have found that a unique mix, including
carotenoid compounds and PUFA-rich phospholipid (such as soy
lecithin, DHA-, EPA- or ARA-rich phospholipid extracts) improves
the bioavailability of carotenoids when consumed by coldwater fish.
Additionally, the phospholipids increase oxidation stability of the
carotenoids compared to other types of standard preparations. It is
well documented that carotenoids are sensitive to photo- and
thermal-oxidation, which results in major carotenoid losses during
feed preparation and storage. Moreover, natural sources of
carotenoids include a high level of saturated oils. Saturated oils
become solidified at low water temperature and thereby reduce
bioavailability of the carotenoid in the animal GI tract. The
present invention overcomes the problems associated with standard
carotenoid formulations by combining carotenoids with PUFA-rich
phospholipid, where the phospholipid increases the efficacy of the
carotenoid absorption at low temperatures.
[0054] The present invention relates broadly to formulations
including carotenoids and PUFA-rich phospholipid compositions.
Additionally, methods for producing such compositions and their use
in formulation of novel feeds are disclosed.
[0055] Examples of phospholipid include, without limitation,
phosphatidyl cholines (such as phosphatidyl choline (PC),
dipalmitoylphosphatidylcholine (DPPC), other disaturated
phosphatidyl cholines), phosphatidyl ethanolamines,
phosphatidylinositol, phosphatidyl serines (sphingomyelin or other
ceramides), various other phospholipids, phospholipid-containing
oils (such as lecithin oils derived from soy beans), or mixtures
and combinations thereof The phospholipids of the present
formulation can also be found in PUFA-rich extracts of single cell
organisms such as, but not limited to, Crypthecodinium sp.,
Schizochytrium sp., Mortierella sp. and Paracoccus sp.
Phospholipids of the present invention can also be derived from
animal sources including, but not limited to, animal organ extracts
(e.g., brain, liver, other animal process wastes), egg yolk, egg
yolk extracts, fish byproducts and fish byproduct extracts (i.e.,
processed waste products from preparation of fish meal or purified
fish oil). Preferred phospholipids are from Crypthecodinium sp.,
Schizochytrium sp. and Mortierella sp., and plant lecithins.
Phospholipids useful for this invention would be those wherein at
least 20% of the fatty acid residues have 2 or more double bonds.
Preferred phospholipids would be those containing at least 20% of
the fatty acid residues with 3 or more double bonds. Particularly
preferred phospholipids would be those containing at least 10% of
the fatty acid residues with 4 or more double bonds. Most
particularly preferred phospholipids would be those containing at
least 20% of the fatty acid residues with 4 or more double
bonds.
[0056] Generally, the weight ratio of carotenoids to PUFA-rich
phospholipid is between about 2:1 and about 1:100, with ratios
between about 2:1 and 1:50 being preferred and ratios between about
1:1 and 1:10 being particularly preferred and ratios between about
1:1 and about 1:5 being especially particularly preferred.
[0057] The effective amount of the carotenoids for use in the
composition of this invention ranges from about 0.1 mg per kg feed
to about 1000 mg per kg feed depending on the carotenoids and the
phospholipid used in the composition. Amounts between about 1 mg
per kg feed to about 500 mg per feed being preferred, with amounts
between about 2 mg per kg feed and 50 mg per feed being
particularly preferred. A sufficient amount of phospholipid is
generally an amount of phospholipid between about 0.01 mg per mg
carotenoids and about 5000 mg per mg carotenoids, with amounts
between about 0.5 mg per mg carotenoids and 2500 mg per mg
carotenoids being preferred, and amounts between 2 mg per mg
carotenoids and about 250 mg per mg carotenoids being particularly
preferred, and amounts between about 2 mg per mg carotenoids and
about 100 mg per mg carotenoids being especially particularly
preferred.
[0058] The compositions of the present invention can be in any
desirable form, including, without limitation, a solid (such as a
powder, granules, a semi-solid such as a paste or the like), an
emulsion, or a solution. An emulsion means that an oil or aqueous
form of the compositions of this invention is emulsified in an
aqueous solution. In addition, the emulsion can be a standard
emulsion or a micro-emulsion where the mixture is forced through a
nozzle or in other methods that generate micro-emulsions. Solutions
of this invention employ a suitable solvent in which the
composition is soluble or highly soluble.
[0059] Generally, the compositions of this invention are formulated
to be directly mixed with other feed ingredients prior to
processing. However, the formulations can also be emulsified or
blended with a carrier oil to top-coat the feed after
processing.
[0060] In formulations of this invention that combine a
phospholipid, such as lecithin, and a carotenoid, such as
astaxanthin, the phospholipid acts to prevent oxidation of the
carotenoids as well as to improve its solubility. Thus, the
formulations of this invention, which supplement carotenoids with
phospholipids, show significantly more stability, thus removing a
major impediment that severely limits the utility of natural
carotenoids in feed preparation. The carotenoid/phospholipid
formulations of this invention not only have increased stability,
but the formulations also increase the bioavailability of the
carotenoids when taken by coldwater animals. Current carotenoid
formulations contain large quantities of high melting temperature
oils. These preparations therefore lose a major part of their
effectiveness when taken by coldwater species due to the phase of
the oil (i.e., solid). The carotenoids of the invention associate
with PUFA-rich phospholipids in such a way as to preserve their
liquidity and become more available for uptake in the small
intestines, especially at low temperatures. Additionally, it is
thought that the PUFA-rich phospholipid-carotenoid formulations of
this invention improve carotenoid bioavailability by interfering
with the interaction of carotenoids with other feed components
during digestion in the fish stomach, permitting carotenoids to
exit the stomach in a bioavailable form.
[0061] For example, the carotenoids (naturally produced by a single
celled-organism or synthetic) can be combined with different
concentrations of either purified phospholipids or crude
phospholipids. For example, PC is available in a purified form
comprising>90% PC or in crude extracts from soybeans in de-oiled
and oiled states (American Lecithin Company). Crude phospholipid
extracts containing over 40% DHA or ARA of total fatty acids are
also available (Advanced BioNutrition Corp., Columbia, Md.). The
presence of PUFA-rich phospholipid, such as lecithin, in the
formulations of this invention prevents carotenoid solidification,
thereby increasing bioavailability of carotenoids in the GI tract
of coldwater species. Thus, the presence of a PUFA-rich
phospholipid in the compositions of this invention allows a
reduction in carotenoid dosages in feed and the shortening of the
administration period prior to harvesting without loosing the
desired coloring.
[0062] Further improvement in bioavailability may be achieved by
the addition of an alkaloid, such as piperine, to the
carotenoid/phospholipid composition.
[0063] The addition of PUFA-rich phospholipids can also
significantly increase the bioavailability of the carotenoids. This
is an improvement, since in certain instances carotenoids have
bioavailabilities of about 50% or less necessitating relatively
large doses of the carotenoids for a longer period of time. The
PUFA-rich phospholipids result in improved bioavailability of the
carotenoids especially by coldwater species. The improved
bioavailability can range from about a 20% increase to as much as
about a 60% or greater increase by carefully choosing the type of
PUFA-rich phospholipid and the ratio of the carotenoids and
PUFA-rich phospholipids.
[0064] It should be noted that a number of substances that are used
as additives to enhance carotenoid absorption are known irritants
or damaging agents of the GI mucosa. Therefore, these would be
contraindicated for use with carotenoids. Such substances would
include: short chain fatty acids (such as citric acid, decanoic
acid, caprylic acid or the like), long-chain unsaturated free fatty
acids (such as oleic acid or the like), detergents (such as BRIJ,
TWEEN-80, sodium deoxycholate, or the like), and chelators of
polyvalent metal cations (such as EDTA, EGTA, or the like).
[0065] Because of their degree of unsaturation, carotenoids are
inherently prone to oxidative degradation. Preserving the integrity
of the double bonds of the carotenoids through processing and
storage is a critical problem in the preparation of feeds, food and
supplements therefore containing such materials. At the same time
the preservation of the double bonds of the carotenoids is critical
for the efficacy of the carotenoid itself. Kyle and Becker (WO
00/54575) have described a process whereby a DHA-containing oil is
stabilized by lecithin at levels up to 8% of the oil. AN additional
aspect of this invention involves the combination of lecithin with
the carotenoid containing material is in the stabilization of the
carotenoid against oxidation.
[0066] Another aspect of the present invention is the combination
of the lecithin with other cellular materials comprising long chain
polyunsaturated fatty acids (LC-PUFAs). Microorganisms such as, but
not limited to, Crypthecodinium, Schizochytrium, Theraustochytrium,
Ulkenia, Mortierella, etc. are prone to oxidation as a result of
their high content of LC-PUFA. Schzochytrium, Thraustochytrium and
Ulkenia, in particular, are very fragile and can release oil during
the process of harvesting and drying. The use of high concentration
of phospholipids (especially lecithin) during the drying process
can impart a high degree of stability to the resulting dry biomass
of these microorganisms and increase the bioavailability of the
LC-PUFAs themselves. Lecithin to biomass ratios from about 1:100 to
about 1:1 are effective in increasing stability and bioavailability
of the oils.
[0067] Methods for Making Carotenoid/Phospholipid Compositions
[0068] One preferred class of compositions of this invention are
compositions that include a carotenoid or carotenoids and PUFA-rich
phospholipid or PUFA-rich phospholipids generally prepared by
contacting carotenoid and phospholipid under conditions to promote
molecular association of the carotenoid and phospholipid. Such
conditions typically will include the use of mixing procedures that
promote molecular interactions and associations, use of a solvent
and/or buffer, and controlled physical parameters (such as
temperature, pressure and time) to permit an optimal degree of
interaction and association.
[0069] The chemical interaction is preferably performed by
aggressive or vigorous mixing. Such mixing procedures include
vortex mixing, other high shear mixing procedures, sonication,
other molecular level mixing procedures, or the like. The time and
temperature of mixing should be designed to maximize interactions
between the carotenoids and the phospholipids without causing
thermal or shear damage to the molecules themselves. Generally, the
mixing time will range from about 5 minutes to several hours, with
times ranging between 10 minutes and 1 hour being preferred.
[0070] Generally, the mixing temperature will range from ambient to
a temperature of at least 10% below the lowest breakdown
temperature for the carotenoids or phospholipids being mixed.
Preferably, the temperature will be between ambient temperature to
about 60.degree. C.
[0071] In preparing the formulations of this invention, the
carotenoids can be mixed with synthetic, purified naturally
derived, or crude phospholipids or can be mixed with various grades
of lecithin or other PUFA-rich oils obtained from single-celled
organisms. Carotenoids may be in the form of pure carotenoid
(synthetic or otherwise) or as cellular material from high
carotenoid microorganisms such as but not limited to Pfaffia or
Heamatococcus and the mixture of phospholipids to microbial cell
biomass may be in the range from 1 part phospholipid to from 1 to
100 parts cellular biomass. Especially useful phospholipid
concentrations range from about 15 to about 93% PC by weight.
Moreover, the formulations can use either de-oiled or oil-based
phospholipid preparations.
[0072] Regardless of the form of the phospholipid, generally the
ratio of carotenoids to phospholipids ranges from about 1:100 to
about 10:1, preferably, from about 1:25 to about 2:1, and
particularly from about 1.0:10.0 to about 1.0: 1.0.
[0073] In formulations using de-oiled phospholipids, the de-oiled
phospholipids are initially dissolved in an organic solvent such as
ethanol, and then mixed with carotenoids. This is followed by
mixing, such as vortexing and/or sonication mixing. In formulations
using oiled phospholipids, the oil-based phospholipids are simply
combined with a carotenoid compound and mixed by vortexing and/or
sonication, if needed. Sonication or mixing temperatures are
preferably between ambient and about 60.degree. C.
[0074] Another preferred process for making the compositions of
this invention includes the dissolving of phospholipids and
carotenoids in a polar solvent. Suitable solvents include, without
limitation, chlorocarbons (such as chloroform, or the like), lower
alcohols (such as methanol, ethanol, isopropanol or the like), or
any other solvent in which the phospholipids and the carotenoids
have some solubility, and the solvent is removable, e.g., by
evaporation, or the like.
[0075] Methods for making LC-PUFA phospholipids compositions. In
preparing the formulations of this invention, the
LC-PUFA-containing biomass such as, but not limited to
Schyzochytrium, can be mixed with synthetic, purified naturally
derived or crude phospholipids or can be mixed with various grades
of lecithin or other PUFA-rich oils obtained from single cell
organisms. Especially useful phospholipids concentrations ranging
from about 15 to about 93% PC by weight. Moreover, the formulations
can use either de-oiled and oiled-based phospholipids preparations.
Mixtures of phospholipids and cellular material containing LC-PUFAs
can range from 1 part to from 1 to 100 parts cellular material.
EXAMPLES
[0076] The following examples are included for example only to
illustrate the preparation of compositions of present invention
containing a carotenoids and PUFA-rich phospholipid, and are in no
way meant to limit the scope or teaching of this invention.
Example 1
Preparation of a Composition of Synthetic Astaxanthin and Soy
Lecithin
[0077] A sample of 60 g of soy lecithin (American Lecithin Co) was
dissolved in ethanol, 30 g synthetic astaxanthin (AHD
International, Atlanta, Ga.) was added, the mixture sonicated at
60.degree. C. for 5 minutes, and the solvent evaporated under
vacuum. The resulting powder can be incorporated with other feed
ingredients or dissolved in oil and top-coated onto the feed
particles.
Example 2
Preparation of a Composition of Haematococcus (Containing Natural
Astaxanthin) and Phospholipid Extract from Crypthecodinium
Species
[0078] A sample of 50 g of algal phospholipids (Advanced
BioNutrition, Columbia, Md.) and 100 g Haematococcus (Naturose,
Cyanotech Corporation Kailua-Kona, Hi.) were mixed vigorously for 1
h at room temperature. The mixture was dissolved in 850 ml of
Menhaden oil (Omega Protein, Houston, Tex.) and used to top-coat
standard fish feed pellets. The feed pellets were top coated at a
level of 20 g of the above mixture per kg feed. This produced a
feed containing about 50 mg astaxanthin per kg feed. This feed was
then used to color the flesh of aquatic animals that consumed the
feed.
Example 3
Preparation of a Composition of Phaffia rhodozyma Yeast Biomass and
Phospholipid Extract from Crypthecodinium sp
[0079] Phaffia yeast was grown under standard conditions in a
fermentor and biomass was harvested by centrifugation and diluted
to 30% solids with water. Then 13.3 g of algal phospholipids (8 g
on a dry weight basis) (ABN, Columbia, Md.) was mixed vigorously
with 333 g of the Phaffia slurry (100 g on a dry weight basis) to
facilitate molecular association between the carotenoid and the
phospholipids. The material was then dried on a rotary drum dryer
at low temperatures and the resulting flakes were milled under
liquid nitrogen to produce a coarse powder. The resulting powder
was then mixed with a commercial trout feed and cold pressed into
feed pellets (1.2-2.0 mm, Ziegler Bros Inc. Gardners, Pa.) using
standard techniques.
Example 4
Preparation of a Composition of Phaffia rhodozyma Yeast Biomass and
Soy Lecithin
[0080] One hundred grams of Phaffia yeast biomass
(Archer-Daniels-Midland Company, Decatur, Ill.) was mixed with
water to give a slurry with a 30% water content. Eight g of soy
lecithin (American Lecithin Co) was added to the slurry and the
resultant mixture was homogenized vigorously to facilitate
molecular association between the carotenoid and the phospholipids.
The slurry was then dried in a freeze dryer and collected as a
powder. This material had the following composition: 1.5%
astaxanthin, 8% phospholipid, 50% fatty acids with 2 or more double
bonds, and 20% of the fatty acids with 4 or more double bonds. This
mixture was then incorporated into 10 kg commercial fishmeal
pellets using standard methods with cold pressing or cold extrusion
(Ziegler Bros Inc. Gardners, Pa.).
Example 5
Feeding of Trout Fish with a Feed Containing Natural Astaxanthin
from Phaffia and a PUFA-Containing Phospholipid
[0081] Five diets were prepared by Ziegler Bros Inc. (Gardners,
Pa.) according to the following compositions:
[0082] Diet 1 contained 12.5 g Phaffia biomass per kg feed (100 mg
astaxanthin/kg feed).
[0083] Diet 2 contained 13.8 g of the composition described in
Example 3 per kg feed (100 mg astaxanthin/kg feed).
[0084] Diet 3 contained 7.6 g of the composition described in
Example 3 per kg feed (50 mg astaxanthin/kg feed).
[0085] Diet 4 contained no Phaffia (0 mg astaxanthin/kg feed).
[0086] Diet 5 contained 7.6 g of the composition described in
Example 4 per kg feed (50 mg astaxanthin/kg feed).
[0087] Five groups of 20 trout fish per group were fed 4.4% body
weight/day for 21 days. White muscle tissues were sampled from 5
fish in each group on day 21 and freeze-dried for 48 h. Total
carotenoids were extracted from the tissues by homogenizing in 5 ml
of absolute ethanol and 5 ml ethyl acetate. The homogenates were
centrifuged (1000.times.g for 5 min) and the supernatants dried
under a stream of nitrogen and dissolved in 2 ml of hexane. Total
carotenoids were measured spectrophotometrically at 470 nm.
[0088] The effect of the diet on muscle pigmentation is presented
in Table 1: TABLE-US-00001 TABLE 1 Absorbance at 470 nm Diet 1 0.19
Diet 2 0.30 Diet 3 0.11 Diet 4 0.05 Diet 5 0.14
[0089] As can be seen from Table 1, Diet 4, with no Phaffia and no
astaxanthin, provided the least amount of muscle pigmentation
indicative of carotenoid content (A.sub.470=0.05). Diet 3 and Diet
5, with no Phaffia and 50 mg astaxanthin provided by the
compositions of Example 3 and Example 4, respectively, provided
intermediate amounts of muscle pigmentation. Diet 1, with Phaffia
biomass providing twice as much, i.e., 100 mg astaxanthin, provided
only a slightly higher amount of coloration than Diets 3 and 5.
Diet 2, with no Phaffia and 100 mg astaxanthin provided by the
composition of Example 3, provided the highest amount of
coloration. It improved the muscle coloring by 56%, compared to
Diet 1.
Example 6
Preparation of Schyzochytrium Biomass with a High Degree of
Oxidative Stability
[0090] Schizochytrium biomass is produced using conventional
fermentation technology and harvested by centrifugal harvesting
processes to a solid content of about 20%. To this 100 g of slurry
(20 g dry weight Schizochytrium containing about 10 g of LC-PUFA
enriched oil) 2 g of soy lecithin (American Lecithin Co.) is added.
The resultant mixture is thoroughly mixed and then dried using a
rotary drum dryer, or any other drying process and collected as
powder of flake. The resulting flake product has a high degree of
oxidative stability and bioavailability relative to a similar
product produced without the lecithin treatment.
[0091] While this invention has been described fully and
completely, it should be understood that, within the scope of the
appended claims, the invention may be practiced otherwise than as
specifically described. Although the invention has been disclosed
with reference to its preferred embodiments, from reading this
description those of skill in the art may appreciate changes and
modification that may be made which do not depart from the scope
and spirit of the invention as described above and claimed
hereafter.
[0092] All references cited herein are incorporated by reference,
including the following.
PATENT REFERENCES
[0093] U.S. Pat. No. 6,261,598
[0094] U.S. Pat. No. 6,476,010
[0095] U.S. Pat. No. 6,436,437
[0096] U.S. Pat. No. 6,403,056
[0097] U.S. Pat. No. 6,358,524
[0098] U.S. Pat. No. 6,296,877
[0099] U.S. Pat. No. 6,413,736
[0100] U.S. Pat. No. 6,022,701
[0101] U.S. Pat. No. 5,972,642
[0102] U.S. Pat. No. 5,935,808
[0103] PA20020177181
[0104] EP-A-0 410 236
[0105] DE-A-12 11 911
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